Lantibiotic mutants and chimeras of enhanced stability and activity, leader sequences therefor, genes encoding the same, and methods of producing and using the same

ABSTRACT

Nisin-subtilin mutant and chimeric pre-peptides were constructed and expressed in a Bacillus subtilis strain that possesses all of the cellular machinery for making subtilin, except for the pre-subtilin gene. The chimera S L  -Nis 1-11  -Sub 12-32  was prepared. This pre-peptide has the subtilin leader sequence (S L ), the N-terminal portion of the structural region derived from nisin and the C-terminal portion of the structural region derived from subtilin. This chimera was accurately and efficiently converted to the mature lantibiotic, as demonstrated by a variety of physical and biological activity assays. In contrast, a S L  -Sub 1-11  -Nis 12-34  chimera was processed into a heterogeneous mixture of products, none of which appeared to be the correct chimeric lantibiotic. The mixture did, however, contain an active minor component with a biological activity that exceeded nisin itself.

This work was supported by National Institutes of Health grant AI24454.Therefore, the U.S. government may have certain rights in the presentinvention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention concerns lantibiotic mutants and chimeras ofenhanced stability and activity, leader sequences for such lantibioticmutants and chimeras, genes encoding such lantibiotic mutants andchimeras (both with and without the leader sequences), and methods ofproducing and using the same.

2. Discussion of the Background

Nisin (a 34-residue long peptide produced by Lactococcus lactis) andsubtilin (a 32-residue long peptide produced by Bacillus subtilis) arethe most thoroughly-studied examples of lantibiotics. Lantibiotics areribosomally-synthesized antimicrobial peptides characterized by thepresence of unusual lanthio and dehydro amino acid residues. Thestructures of nisin and subtilin are shown in FIG. 1. Their biosynthesisinvolves several post-translational modifications; e.g., dehydration ofserines and threonines, formation of thioether crosslinkages betweendehydro residues and cysteines, translocation, removal of a leadersequence, and/or release of the mature antimicrobial peptide into theextracellular medium (reviewed in refs. 1-3 below).

Gene-encoded antimicrobial peptides constitute a family of naturalproducts whose known members are expanding rapidly in number anddiversity, and are produced by many kinds of organisms, ranging frombacteria to eukaryotes, including mammals (1, 4-6). The ubiquity ofanti-microbial peptides among widely diverged organisms implies that thepeptides have been subject to many different strategies for achievingtheir antimicrobial properties, some of which are quite different fromthe properties and corresponding mechanisms of classical antibioticssuch as penicillin. It may therefore be possible to supplement thearsenal of therapeutic antimicrobial agents that has been depleted as aresult of the evolution of resistance among microbes.

An advantage unique to gene-encoded antimicrobial peptides is that theirstructures can be readily manipulated by mutagenesis, which provides afacile means for constructing and producing the large numbers ofstructural analogs needed for structure-function studies and rationaldesign. Whereas this advantage is shared by all gene-encodedantimicrobial peptides, the lantibiotics are unique in possessing theunusual dehydro and lanthio amino acid residues, which are absent frommagainins (7-9), defensins (10-13), or cecropins (14, 15). This meansthat the lantibiotics offer chemical and physical properties, and hencebiological activities, that are not attainable by polypeptides that lackthese residues.

For example, the dehydro residues (dehydroalanine, or "Dha," anddehydrobutyrine, or "Dhb") are electrophiles, whereas none of the 20common natural amino acids is electrophilic. The thioether crosslinkagesof lantibiotics are more resistant to cleavage or breakage than the morecommon disulfide bridge of proteins lacking lanthio residues. Forexample, a thioether crosslinkage can survive reducing conditions andextremes of pH and temperature better than a disulfide bridge (16).

A concern when making mutants of lantibiotics is the effect of themutations on the post-translational modification process, because amutation that disrupts processing makes the biosynthesis of thecorresponding mature lantibiotic peptide impossible. All knownlantibiotic prepeptides contain an N-terminal region that is cleavedduring maturation. For the Type A lantibiotics (e.g. nisin, subtilin,epidermin), this leader region is highly conserved (17). Participationof the leader sequence in the orchestration of post-translationalmodification and secretion has been proposed (17, 18).

Certain mutations in the leader region of the nisin prepeptide haverendered the cell incapable of nisin production (19), whereas manymutations in the structural region of several lantibiotics do notdisrupt processing (e.g., U.S. application Ser. No. 07/981,525 and refs.20 and 21)). When the complete nisin prepeptide consisting of the nisinleader region and the nisin structural region (N_(L) -Nis₁₋₃₄) wasexpressed in a subtilin-producing cell, no nisin-related peptideproducts were detectable (22, 23). However, when a chimera consisting ofthe subtilin leader region and the nisin structural region (S_(L) -Nis₁₋₃₄) was expressed in a subtilin-producing cell, an inactivenisin-like peptide was produced in which the leader region had beencorrectly cleaved and which contained a full complement of unusual aminoacids (22). The lack of activity was attributed to the formation ofincorrect thioether crosslinkages (22).

Similarly, when a prepeptide consisting of a subtilin leader region anda nisin structural region was expressed in a nisin-producing cell, thenisin structural region contained the unusual amino acids, but theleader was not cleaved (24). It has also been reported that expressionof a prepeptide consisting of the nisin structural region fused to asubtilin-nisin chimeric leader region, S_(L)(1-7) -N_(L)(8-23)-Nis.sub.(1-34), forms active nisin when expressed in asubtilin-producing cell (23).

These results imply that subtilin processing strains such as B. subtilisare not capable of recognizing the nisin prepeptide (which is ordinarilyexpressed in Lactococcus lactis) and converting it to nisin. However,the subtilin processing machinery will perform modification reactions onthe nisin structural peptide if it is attached to a subtilin leaderregion, although the modifications seem to be misdirected so that activenisin is not always produced. Finally, the subtilin processing machinerywill produce active nisin if the leader region is an appropriatecombination of subtilin leader and nisin leader sequences.

Lantibiotics are known to be useful bacteriocides and foodpreservatives. Methods of producing lantibiotics are also known.Lantibiotics offer the advantages of peptide products, in that they aremore easily digested, tolerated and/or secreted by humans, other mammalsand other animals which may ingest the same than are some so-called"small molecule" preservatives. Therefore, a need is felt for newlantibiotics having improved chemical, physical and/or biologicalproperties and for improved methods of producing the same.

SUMMARY OF THE INVENTION

The present invention concerns polynucleic acids which encode a chimericor mutant lantibiotic of the formula:

    (leader)-(lantibiotic)

where the leader is selected from the group consisting of the subtilinleader sequence, the nisin leader sequence, and chimeras of saidsubtilin leader sequence and said nisin leader sequence which permitproduction of an active lantibiotic in a lantibiotic-producing host, andthe lantibiotic is a mutant or chimeric lantibiotic, preferably ofsubtilin and/or nisin; vectors and plasmids containing the same;transformants containing the same, capable of expressing a prepeptideand/or biologically active peptide from the same; prepeptides encoded bythe polynucleic acids; biologically active peptides expressed and/orprocessed by lantibiotic-producing hosts; methods of making thepolynucleic acids, vectors, plasmids, prepeptides and biologicallyactive peptides; and methods of using the same.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structures of nisin (SEQ ID NO:1) and subtilin (SEQ IDNO:2) as determined by Gross and co-workers (37-39); the unusual aminoacids Aba (aminobutyric acid), Dha (dehydroalanine), Dhb(dehydrobutyrine or β-methyldehydroalanine), Ala-S-Ala (lanthionine) andAba-S-Ala (β-methyllanthionine) were introduced by post-translationalmodifications as described hereinbelow;

FIG. 2 shows a strategy for construction of nisin-subtilin chimeras;

FIG. 3 is an HPLC chromatogram of the Nis₁₋₁₁ -Sub₁₂₋₃₂ chimeraconstructed as shown in FIG. 2;

FIG. 4 shows the resolution of the Nis₁₋₁₁ -Sub₁₂₋₃₂ chimera into twoforms on the HPLC column (inset), resulting in the appearance of a newpeak; the Nis₁₋₁₁ -Sub₁₂₋₃₂ chimera was constructed as shown in FIG. 2and expressed and isolated as described below except that the cells weregrown for a longer time (into the stationary phase);

FIG. 5 shows the NMR and mass spectra of the Early Peak and Late Peak,as defined in the HPLC elution profiles in FIG. 4;

FIG. 6 shows the NMR spectra of the Nis₁₋₁₁ -Sub₁₂₋₃₂ chimera during thecourse of a 72-day incubation, demonstrating the stability of thechimera; and

FIG. 7 shows HPLC profiles and mass spectra of the Sub₁₋₁₁ -Nis₁₂₋₃₄chimera.

DETAILED DESCRIPTION OF THE INVENTION

The present invention explores the contribution of the structural regionand its relationship to the leader region by the construction andexpression of nisin-subtilin chimeras which contain chimericnisin-subtilin structural regions fused to the subtilin leader region.The present inventors have discovered that chimeras in which theC-terminal portion of the structural region correspond to subtilin areprocessed correctly and give active products, whereas those in which theC-terminal portion of the structural region corresponds to nisinproduces a heterogeneous mixture of products, most of which, but notall, are inactive.

The phrase "polynucleic acid" refers to RNA or DNA, as well as mRNA andcDNA corresponding to or complementary to the RNA or DNA isolated from alantibiotic-producing host. The term "gene" refers to a polynucleic acidwhich encodes a peptide, prepeptide, protein or marker, or to a vectoror plasmid containing such a polynucleic acid.

In the present application, a "chimera" refers to a peptide or proteinin which the amino acid sequence is taken in part from a first peptideor protein, and in part from a second, distinct protein or peptide. A"mutant" gene or peptide refers to a gene or peptide having a sequencewhich differs from the corresponding naturally-occurring sequence inthat one or more bases or residues are deleted, substituted or added atany position therein, including either terminus.

In the present application, the following formulaic indicators have thefollowing meanings:

    ______________________________________    S.sub.L :  the subtilin leader sequence    S.sub.L(x-y) :               the subtilin leader sequence from position x               to position y    N.sub.L :  the nisin leader sequence    N.sub.L(x-y) :               the nisin leader sequence from position x to               position y    Sub.sub.x-y :               the sequence of the subtilin peptide from               position x to position y    Nis.sub.x-y :               the sequence of the nisin peptide from               position x to position y    ______________________________________

In the context of the present application, the chimera "Nis₁₋₄ Sub₅₋₃₂ "is the same as the chimera "Nis₁₋₁₁ Sub₁₂₋₃₂ " since the amino acids atpositions 5-11 of both nisin and subtilin are identical. Thus, forexample, "Nis₁₋₇ Sub₈₋₃₂ " is the same as each of Nis₁₋₄ Sub₅₋₃₂ andNis₁₋₁₁ Sub₁₂₋₃₂.

The "lantibiotic processing machinery" refers to the metabolic eventsoccurring in a lantibiotic-producing microorganism which result inprocessing and formation of the lantibiotic. For example, the "subtilinprocessing machinery" and the "nisin processing machinery" refer tothose metabolic processes and events occurring, respectively, in asubtilin-producing microorganism which result in the processing and/orformation of subtilin, and in a nisin-producing microorganism whichresult in the processing and/or formation of nisin.

Naturally-occurring nisin and subtilin, leader sequences and genesencoding the same are disclosed in U.S. application Ser. No. 07/214,959,now U.S. Pat. No. 5,218,101, incorporated herein by reference in itsentirety. Subtilin mutants and methods of producing and using the sameare described in U.S. application Ser. Nos. 07/981,525 and 08/220,033,each of which is incorporated herein by reference in their entireties.

In the present application, "biological activity" preferably refers toactivity against Bacillus cereus spores and/or vegetative cells.Preferably, biological activity against Bacillus cereus spores ismeasured using the "halo assay" described in the experimental sectionhereunder, and biological activity against Bacillus cereus vegetativecells is measured using the liquid culture assay described in theexperimental section hereunder.

The present invention concerns polynucleic acids which encode a chimericor mutant lantibiotic of the formula:

    (leader)-(lantibiotic)

where the leader is selected from the group consisting of the subtilinleader sequence, the nisin leader sequence, and chimeras of saidsubtilin leader sequence and said nisin leader sequence which permitproduction of an active lantibiotic in a lantibiotic-producing host, and

the lantibiotic is a mutant or chimeric lantibiotic, preferably ofsubtilin and/or nisin;

subject to the proviso that when the lantibiotic is a mutant subtilin inwhich the 4-position of native subtilin as shown in FIG. 1 issubstituted with isoleucine and in which the 5-position may besubstituted with alanine, the leader is not the subtilin leadersequence.

The present polynucleic acid may encode a chimeric leader sequence ofthe formula:

    S.sub.L(1-x) -N.sub.L( x+1!-23)

or

    N.sub.L(1-x) -S.sub.L( x+1!-23)

where x is a number of from 1 to 22, selected such that the lantibioticprocessing machinery of a lantibiotic-producing host transformed withthe present gene produces either a biologically active lantibiotic or aprepeptide which can be converted to a biologically active lantibioticusing the lantibiotic processing machinery of an appropriatelantibiotic-producing host. (When x is 1, "1-x" becomes 1, and when x is22, " x+1!-23" becomes 23.) When a chimeric leader is used, x ispreferably from 5-18, more preferably from 6-15, and most preferably,the chimeric leader is S_(L)(1-7) -N_(L)(8-23).

However, the present polynucleic acid preferably encodes anaturally-occurring lantibiotic leader sequence, such as S_(L) or N_(L).

Preferably, the lantibiotic-producing host transformed with the presentpolynucleic acid is a subtilin-producing host or a nisin-producing host.More preferably, the subtilin-producing host is a strain of Bacillussubtilis, such as B. subtilis 6633 or B. subtilis 168, and thenisin-producing host is a strain of Lactobacillus lactis, such as L.lactis 11454. Most preferably, when a subtilin-producing host is used toproduce or express the present peptide or prepeptide, the polynucleicacid encodes either the S_(L) leader sequence or a S_(L)(1-x) -N_(L)(x+1!-23) chimeric sequence where x is 7, and when a nisin-producing hostis used to produce or express the present peptide or prepeptide, thepolynucleic acid encodes the N_(L) leader sequence.

Preferably, the lantibiotic encoded by the present gene and processed bya lantibiotic-producing host is one of the formula: ##STR1## where Xaais any amino acid, including a dehydro amino acid residue or one of thetwo residues of a lanthio amino acid as defined herein, and Z is eitherNis₁₃₋₃₄ or Sub₁₃₋₃₂, with the proviso that when Z is Sub₁₃₋₃₂, then,simultaneously, the 1-position is not Trp, the 2-position is not Lys,the 4-position position is not Ile, and the 5-position is not Dha orAla.

Preferred residues at the 1-position include Trp and Ile; at the2-position include Lys and Dhb; at the 4-position include Ile; at the5-position include Dha and Ala; and at the 12-position include Val andLys; and conservatively substituted forms thereof. An amino acid residuein a protein, polypeptide, or prepeptide is conservatively substitutedif it is replaced with a member of its polarity group as defined below:

Basic amino acids:

lysine (Lys), arginine (Arg), histidine (His)

Acidic amino acids:

aspartic acid (Asp), glutamic acid (Glu), asparagine (Asn), glutamine(Gln)

Hydrophilic, nonionic amino acids:

serine (Ser), threonine (Thr), cysteine (Cys), asparagine (Asn),glutamine (Gln)

Sulfur-containing amino acids:

cysteine (Cys), methionine (Met)

Aromatic amino acids:

phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp)

Hydrophobic nonaromatic amino acids:

glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine(Ile), proline (Pro)

Dehydro amino acids:

dehydroalanine (Dha), dehydrobutyrine (Dhb)

Lanthio amino acids:

lanthionine (Ala-S-Ala), β-methyllanthionine (Aba-S-Ala),β,β'-dimethyllanthionine (Aba-S-Aba)

Particularly preferred residues at the 1-position include Trp and Ile;at the 2-position include Lys and Dhb; at the 4-position include Ile; atthe 5-position include Dha; and at the 12-position include Val and Lys.

Based on the experimental results described herein, it is expected thatany amino acid can exist at positions 1, 2, 4 and 12 of the maturelantibiotic, and a biologically active peptide can be produced. Theprocedures described herein easily enable one to produce any such mutantor chimeric peptide, then test its biological activity against B. cereusspores and/or cells. As one can easily tell in comparing the sequencesof mature nisin and mature subtilin, the differences at positions 1, 2,4 and 12 are a result of non-conservative substitutions. Thus, itappears that the specific identities of the residues in the nativeproteins may not be essential to maintaining a high degree of biologicalactivity, and any amino acid residue at any one of these positions,particularly where the activity is the same as or equal to that ofsubtilin or nisin, is expected to be producible and useful.(Naturally-occurring nisin and subtilin, and polynucleic acids encodingthe same, are excluded from the scope of the present invention.)

The present invention also concerns an expression vector, plasmid andtransformant comprising the present polynucleic acid. In preferredembodiments, the expression vector is one in which the presentpolynucleic acid is inserted into the BstEII-BstEII site of the plasmidpSMcat (deposited under the terms of the Budapest Treaty at the AmericanType Culture Collection, Rockville, Md. 20852, U.S.A., under DesignationNo. 75914; also see Ser. No. 07/981,525) or the SpeI-BstEII site ofpACcat (see the description below).

The present invention also concerns a method of producing apolynucleotide encoding a mutant or chimeric lantibiotic prepeptide,comprising (A) replacing a native gene encoding a lantibiotic with agene consisting essentially of a selective marker, such that alantibiotic-producing host in which the native lantibiotic gene isreplaced is unable to produce the lantibiotic (as determined by a haloor liquid culture assay), and (B) subsequently replacing the selectivemarker with a polynucleic acid encoding the mutant or chimericlantibiotic. The polynucleotide encoding a mutant or chimericlantibiotic prepeptide may be in the form of a vector or plasmid, whichmay have appropriate sequences and an appropriate construction toexpress the mutant or chimeric peptide or prepeptide.

When a suitable lantibiotic-producing host is transformed with thepolynucleotide encoding the mutant or chimeric lantibiotic prepeptide,only the mutant or chimeric lantibiotic is produced in significantamounts by the transformant.

Some or all mutant or chimeric lantibiotics produced by suchtransformants are expected to exhibit biological activity. The mutant orchimeric lantibiotic produced by the present method preferably has abiological activity (as defined above) equal to or greater than that ofnisin, more preferably, at least twice that of nisin, and may even havea biological activity of from 4 times to 35 times that of nisin (see theexperimental section below). The mutant or chimeric lantibioticsproduced by such transformants may also exhibit improved chemicalstability, as measured by the disappearance of the signals of thevinylic protons of the dehydro residues in ¹ H NMR spectra as a functionof time. Preferably, the mutant or chimeric lantibiotics produced by thepresent method and/or transformants exhibit a half-life of at least 48days, more preferably at least 72 days, and most preferably, a half-lifewhich cannot be determined by ¹ H NMR spectroscopy after 72 daysincubation in the dark in aqueous solution.

The present method of producing a mutant or chimeric lantibiotic alsoinvolves the step of culturing a lantibiotic-producing host transformedwith the present polynucleic acid, vector or plasmid in a suitablemedium, and recovering the lantibiotic from the culture medium.Culturing is generally performed for a length of time sufficient for thetransformant to produce, process and/or secrete the mutant or chimericpeptide. A "suitable" medium is one in which the lantibiotic-producingmicroorganism grows and produces the lantibiotic peptide or prepeptide.This method may also include the step of rupturing or lysing thetransformed cells prior to recovering the lantibiotic. Alternatively,the cells of the transformed host may be recovered and recultured.

Continuous processes for producing the present lantibiotic are alsoenvisioned, comprising the additional steps of withdrawing the culturemedium continuously or intermittently, separating the transformant fromthe withdrawn culture medium, recirculating the separated transformantto the culture vessel, and recovering the lantibiotic from the withdrawnculture medium from which the transformant has been separated.

The present invention also concerns a method of treating, killing orinhibiting the growth of microorganisms and/or spores thereof,comprising contacting a microorganism, spore thereof or a medium subjectto infection or infestation by said microorganism or spore, with aneffective amount or concentration of the present lantibiotic mutant orchimera. In this context, microorganisms and/or spores to be treated bythis method are those which are killed or whose growth is inhibited by alantibiotic. An "effective amount or concentration" refers to an amountor concentration which kills or inhibits the growth of the microorganismor spore.

Any use for which nisin, subtilin or other known lantibiotics are usedare also envisioned for the present lantibiotic. For example, a mediumwhich can be treated by this method may be a food product or a substancewhich is used in making food products. Furthermore, the medium may be aninert carrier, and such a composition may be used in a conventionalmanner as a bacteriocide.

Other features of the invention will become apparent in the course ofthe following descriptions of exemplary embodiments which are given forillustration of the invention and are not intended to be limitingthereof.

EXAMPLES

Bacterial strains, cloning vectors, and mutagenesis. Bacillus subtilis168 strains and cloning vectors used were LHermΔS (deposited under theterms of the Budapest Treaty at the American Type Culture Collection,Rockville, Md. 20852, U.S.A., under the ATCC Designation No. 55625; seealso U.S. application Ser. No. 07/981,525 and ref. 21), pTZ19U (LifeTechnologies, Gaithersberg Md.), pSMcat (see U.S. application No.07/981,525 and ref. 21), and pACcat (this work). Structural mutants ofsubtilin were constructed and expressed using the cassette mutagenesissystem previously described in U.S. application Ser. No. 07/981,525(also see ref. 21). Cloning vector pACcat was constructed by replacingthe upstream BstEII site in the plasmid pSMcat with an SpeI site bymutagenesis, thus making the downstream BstEII site a unique site.

Synthetic oligonucleotides were annealed, filled in using Klenowfragment, restricted with EcoRI and HindIII and cloned into theEcoRI-HindIII site of pTZ19U. The cloned fragment, which contained themutation, was cut out with appropriate restriction enzymes and clonedinto the corresponding site of the pSMcat vector or the pACcat vector.The mutated sequence was conf irmed by sequence analysis of the clonedinsert using the SEQUENASE version 2.0 sequencing kit from United StatesBiochemicals (Cleveland, Ohio.). This mutant gene was introduced intothe chromosome of LHermfΔS (from which the natural subtilin gene hadbeen deleted) by transformation and Campbell-type integration usingselection on chloramphenicol plates (21).

Culture conditions and purification of chimeric peptides. Strainsproducing the mutant peptides were grown in Medium A (21, 25), modifiedto contain 2% sucrose and 10 μg/mL chloramphenicol. The culture wasincubated with vigorous aeration for 25-35 hr at 35° C., acidified to pH2.5 with phosphoric acid and heated to 121° C. for 3 min to inactivateproteases. A 0.5 part by volume of n-butanol (relative to 1 part byvolume of culture) was added. The mixture was stirred at 4° C. for 2 h,allowed to stand at 4° C. for 2 h, and centrifuged. Acetone (2.5 partsby volume, relative to 1 part by volume of the mixture) was added to thesupernatant, and the resultant mixture allowed to stand at -20° C. for16 h, and centrifuged. The pellet was lyophilized and resuspended in 20%acetonitrile with 0.05% trifluoroacetic acid. This suspension wasimmediately purified on a C-18 reverse-phase-HPLC column using atrifluoroacetic acid (0.05%) -water-acetonitrile gradient in which theacetonitrile varied from 0 to 100% over 30 min at a rate of 1.2 mL/min,unless indicated otherwise.

NMR and mass spectral analyses. Samples for ¹ H NMR spectral analysiswere dissolved in deuterated water (99.96 atom % D, Aldrich ChemicalCo.), lyophilized (repeated twice) to exchange protons and dissolved inD₂ O to a final concentration of 2-3 mg/mL. ¹ H NMR spectra wereobtained using a Bruker AMX-500 spectrometer interfaced to an Aspect3000 computer. Spectra were obtained at a constant temperature of 295K,using selective solvent suppression. Data were processed using UXNMRsoftware. Mass spectral analysis was performed by PeptidoGenic Research& Co. (Livermore, Calif.) on a Sciex API I Electrospray MassSpectrometer which has an analysis range of over 200,000 Da with+/-0.01% accuracy, on 5 μL samples at a concentration of about 5pmol/μL. The reported masses are those calculated as the most probablevalues based on the different m/z forms.

Measurement of biological activity. Biological activity was measuredusing an inhibition zone (halo) assay (ref. 21 and U.S. application Ser.No. 07/981,525) and a liquid culture assay (26). HPLC fractions weretested for activity by spotting 15 μL onto an agar plate (modifiedMedium A), incubating at 37° C. for 15 min., spraying with B. cereus Tspores and incubating at 37° C. for 16 hr. Positive inhibition isdefined as and was determined by a clear zone containing spores thatwere inhibited during outgrowth surrounded by an opaque lawn of cellsderived from the spores that had become vegetative.

In the liquid culture assay, various concentrations of peptide wereadded to a suspension of B. cereus T spores in modified medium A andincubated in a rotating drum shaker at 30° C. for 90 min. The inhibitoryeffects were evaluated using phase contrast microscopy and aKlett-Summerson calorimeter. After incubation, cells were viewed byphase-contrast microscopy to determine their stage of outgrowth. Thosecells in early stages of outgrowth (phase-dark and swollen, but onlyslightly elongated) were considered inhibited. Those cells that werefully elongated and/or divided were considered to be not inhibited. Theinhibitory concentration is the concentration of peptide which arrests amajority of the population of spores at the stage of early outgrowthafter the 90-min incubation period. Further, spore germination andoutgrowth are accompanied by known changes in the optical density at 650nm. Thus, the stage of outgrowth can be monitored and the inhibitoryconcentration can be confirmed using a Klett-Summerson colorimeter.Definitive determinations of the inhibitory concentration of peptideadvantageously employ both phase-contrast microscopy and measurement ofthe optical density at 650 nm.

Relative amounts of peptide were also estimated by integration of peakareas (measured at 214 nm) of the HPLC profiles, using nisin as astandard. It was assumed that the extinction coefficients of the mutantpeptides are the same as nisin at this wavelength.

The activity of the chimeric Nis₁₋₁₁ -Sub₁₂₋₃₂ peptide towardsinhibiting B. cereus vegetative cells was also determined. Heat-shockedB. cereus T spores (150 micrograms) were added to 1% tryptone(Difco)-0.1M Tris-phosphate buffer (2 ml) at pH 6.8. The mixture wasincubated for 2 hours at 37° C. in a rotating drum shaker, whereupon allof the spores were in the vegetative state. The chimeric peptide wasthen added, and incubation was continued for one additional hour. Celllysis was monitored by turbity (measured in Klett units), and theintegrity of the cell was determined by phase-contrast microscopy.Relative inhibitory effectiveness was measured as the amount ofinhibitory peptide required to reduce the turbity by 50%.

SDS-PAGE analysis. The sizes of the peptides were estimated usingTRICINE-sodium dodecylsulfate polyacrylamide gel electrophoresis,designed for proteins in the range of from 1 to 100 kD (27), using a 4%stacking gel, a 10% spacer gel, and a 16.5% separating gel. Gels weresilver-stained using KIT #161-0443 from Bio-Rad (Richmond, Calif.)according to manufacturer's instructions.

RESULTS

Inspection of the structures of nisin and subtilin (shown in FIG. 1)reveals that the number and locations of the thioether rings and Dharesidues are conserved. Each peptide has one Dhb residue, but itsposition is not conserved. The N-terminal region is relativelyconserved, except for 3 nonconservative differences out of the first 11residues. Nisin has isoleucine at position 1, whereas subtilin has abulky aromatic tryptophan. Subtilin has a positively-charged lysine atposition 2, whereas nisin has an unusual Dhb residue. Finally, subtilinhas a negatively-charged glutamate at position 4, whereas nisin has aneutral aliphatic isoleucine.

In previous work, the Glu₄ of subtilin was changed to the Ile₄ of nisin,and a mutant with enhanced chemical stability and activity was obtained(ref. 21 and U.S. application Ser. No. 07/981,525). The presentinvention evolved from changing the other two residues at positions 1and 2 to give a subtilin analog with a nisin-like N-terminus. Thisanalog would have only hydrophobic residues at the N-terminus, as wellas a fourth dehydro residue at a location that is unfamiliar to thesubtilin processing machinery of B. subtilis. If the subtilin-producingorganism is unable to process such a mutant protein/gene properly, theentire processing pathway could abort. Since the subtilin machinery ofB. subtilis cannot process the S_(L) -Nis₁₋₃₄ prepeptide to an activeproduct (22), it is difficult to predict how the subtilin processingmachinery would handle the S_(L) -Nis₁₋₁₁ -Sub₁₂₋₃₂ prepeptide.

Construction and expression of the Nis₁₋₁₁ -Sub₁₂₋₃₂ chimera. Using themutagenesis strategy shown in FIG. 2, residues 1, 2, and 4 in thesubtilin structural region were changed to those of nisin. A mutagenesiswas performed in the plasmid pSMcat, a cassette-mutagenesis plasmid thatcontains a copy of the subtilin structural gene upstream from a cat gene(U.S. application Ser. No. 07/981,525 and ref. 21). When this plasmid istransformed into the B. subtilis 168 host LHermΔS and selected onchloramphenicol, the subtilin gene is integrated into the chromosomalsubtilin (spa) operon (U.S. application Ser. No. 07/981,525 and ref. 21)at the site from which the natural subtilin gene has been deleted. Thesequence of the Nis₁₋₁₁ -Sub₁₂₋₃₂ chimera (SEQ ID NO:5) and thenucleotide sequence (SEQ ID NO:4) that encodes it is shown (top), inwhich the 32-residue mature Nis₁₋₁₁ -Sub₂₋₃₂ sequence is numbered.Immediately below are the mutagenic oligonucleotides (SEQ ID NO:6) and(SEQ ID NO:7) used to construct this sequence. The sequence (SEQ IDNO:8) of the Sub₁₋₁₁ -Nis₁₂₋₃₄ chimera (SEQ ID NO:9) and theoligonucleotides (SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12) used toproduce it are shown at the bottom.

The Nis₁₋₁₁ -Sub₁₂₋₃₂ chimeric gene was integrated into the chromosomeof B. subtilis LHermΔS as described above and cultured as describedabove. The expressed polypeptide products were isolated from theextracellular fluid and subjected to HPLC chromatography. Samples werecollected at 1-min intervals and assayed for activity using the haloassay described herein. FIG. 3 shows the HPLC elution profile of thepeptides isolated from cells in early stationary phase. A single largepeak emerged from the column, and it possessed antimicrobial activity.Electrophoresis on TRICINE (polyacrylamide)-SDS gels and silver-stainingshowed a single major band with a relative molecular mass between 3,000and 3,200, consistent with the predicted molecular weight of the Nis₁₋₁₁-Sub₁₂₋₃₂ chimera.

The major peak contained the only activity. In FIG. 3, the stained gelis shown in a panel beside the peak (sample, left lane; size standard,right lane). Standards shown in the stained gel are 2.5 kD myoglobinfragment (F3); 6.2 kD myoglobin fragment (F2); and the 8.1 kD myoglobinfragment (F1). The expected mass of the Nis₁₋₁₁ -Sub₁₂₋₃₂ chimera is3186 Da, which is consistent with the position of the band in the samplelane.

Appearance of a succinylated form of the Nis₁₋₁₁ -Sub₁₂₋₃₂ chimeraduring late growth stages, as determined by proton NMR and mass spectralanalysis. In an attempt to attain a higher yield of material, theculture was allowed to incubate into late stationary phase, whereuponthe HPLC column profile showed two peaks, one being the original peak,with a second peak trailing slightly behind (inset, FIG. 4). Hereafter,the first peak is called the "Early Peak," and the second peak is calledthe "Late Peak." The two peaks ("Early Peak and "Late Peak") werecollected separately and subjected to NMR spectroscopy as shown in FIG.5. The spectra show that the Late Peak is contaminated by the EarlyPeak. Using the 0-100% acetonitrile gradient described above, thesepeaks were separated by only 1 min. Consequently, the two expressedforms of the chimera were chromatographed using a shallower gradient(35-60% acetonitrile over 45 min; center, FIG. 4), whereupon the EarlyPeak and Late Peak were separated by 4 min. Further experiments wereperformed using the more purified material.

The results of halo assays are shown above the center HPLC profile, witharrows indicating the positions in the profile from which the samplesused for halo assays had been derived. The antimicrobial activity isassociated mainly with the Early Peak. The halo assays in FIG. 4 do notdetect any activity in the Late Peak, but when higher concentrationswere tested, its activity was found to be about 10-fold lower than theEarly Peak (data not shown). This is reminiscent of the observation thatB. subtilis 6633 (the natural producer of subtilin) and LH45 (asubtilin-producing derivative of B. subtilis 168) produce two forms ofsubtilin (28). When B. subtilis 6633 is incubated into late stationaryphase, there is an accumulation of subtilin that has been succinylatedat its N-terminus (29). The succinylated subtilin is significantly lessactive than the normal unsuccinylated subtilin.

The Late Peak was therefore suspected to be the succinylated form of theEarly Peak. This was confirmed by mass spectral analysis (FIG. 5),showing that the Early Peak consists mainly of a species with anMr=3185.98 (panel A), which conforms exactly to the calculated mass of3185.98 Da expected for the mature Nis₁₋₁₁ -Sub₁₂₋₃₂ chimera. The LatePeak gave a mass of 3286.78 Da (panel B), which is consistent with acalculated mass of 3286.78 Da, corresponding exactly to the 100 Daincrease expected from addition of a succinyl group to the matureNis₁₋₁₁ -Sub₁₂₋₃₂ chimera.

In order for these expected masses to occur, it is necessary for thechimeric prepeptides to have undergone the full panoply ofpost-translational modifications in which 8 serines and threonines aredehydrated, 5 thioether crosslinkages are formed, and the leader regionis cleaved at the proper residue. The NMR spectrum of a mixture of theEarly Peak and Late Peak is shown in panel C. Resonances shifted by thepresence of the succinyl group are identified by asterisks (Dhb₂ *, Dha₅*).

The NMR spectra of the Early Peak (panel D) and of the mixture (panel C)of the Early and Late Peaks show resonances that correspond to the Dhb₂and Dha₅ resonances contributed by the nisin part of the molecule and tothe Dhb₁₈ and Dha₃₁ residues contributed by the subtilin portion of themolecule. Identification of the Dhb₂, Dha₅, Dhb₁₈, and Dha₃₁ peaks wasby correlation with NMR spectra obtained previously for nisin (33) andsubtilin (U.S. application Ser. No. 07/981,525 and ref. 21).

Succinylation of subtilin has been shown to cause a shift in theresonance of the Dha₅ residue (29), attributable to a change in thechemical environment of Dha₅ caused by the presence of the N-terminalsuccinyl group. Since the Dhb₂ residue in the succinylated chimera iseven closer to the succinyl group, a shift in its resonance would beexpected. The spectrum shown in panel C, which includes resonances ofthe succinylated chimera, confirms these expectations, and shows ashifted resonance for Dha₅ (labeled as Dha₅ *), and for Dhb₂ (labeled asDhb₂ *). Succinylation of the Nis₁₋₁₁ -Sub₁₂₋₃₂ chimera in the samemanner as subtilin also means that the cell treats the chimera in acompletely normal way, and that the succinylation system must be able totolerate the differences in the N-terminal end of the chimera.Consequently, it appears that at least the 5 N-terminal residues of themature, processed peptide are not critical for recognition by theprocessing machinery.

The biological activity of the Nis₁₋₁₁ -Sub₁₂₋₃₂ chimera. Nisin andsubtilin can inhibit spore-forming food-spoilage bacteria fromundergoing outgrowth from spores to the vegetative state, as well asinhibit cells that are in the vegetative state (30). The mechanism ofinhibition of these types of cells is different, as it has been shownthat the Dha, residue is critical for subtilin to inhibit sporeoutgrowth, but not for subtilin to inhibit vegetative cells (31).

The activity of the two purified forms of Nis₁₋₁₁ -Sub₁₂₋₃₂ weretherefore measured against outgrowing spores and vegetative cells, andcompared to nisin. Since the activities of subtilin and E4I-subtilinhave previously been compared to nisin (21), the relative activitiesamong all these forms can be inferred in terms of relative nisin units.The activity of Nis-₁₋₁₁ Sub₁₂₋₃₂ against spore outgrowth was estimatedby the halo assay and the liquid assays, and against vegetative cells bythe liquid assay.

The Nis₁₋₁₁ -Sub₁₂₋₂₁ chimera was active against both spore outgrowthand vegetative growth. The specific activities of the chimera and nisinwere so similar that they could not be distinguished in either theirability to inhibit spore outgrowth or to inhibit vegetative cells (datanot shown). Accordingly, one sees inhibition of spore outgrowth at about0.2 μg/ml, and against vegetative cells at about 2 μg/ml, with both thechimera and nisin. Based on previous measurements (21, 26, 31), thismeans that the Nis₁₋₁₁ -Sub₁₂₋₃₂ chimera is about 2-fold more activethan E4I-subtilin, and about 6-8 times more active than naturalsubtilin.

Stability of the dehydro residues in the Nis₁₋₁₁ -Sub₁₂₋₃₂ chimeraduring incubation in aqueous solution. The chemical and biologicalinstability of subtilin have been correlated with the tendency ofresidue Dha₅ to spontaneously undergo chemical modification whichresults in disappearance of the Dha₅ peak in the NMR spectrum, and lossof activity against spores (21, 28, 32). This instability of residueDha₅ has been attributed to the participation of the carboxyl group ofthe Glu₄ residue of subtilin in the modification process. Accordingly,changing Glu₄ to Ile₄ (E4I-subtilin) dramatically enhances the chemicalstability of the Dha₅ residue (U.S. application Ser. No. 07/981,525 andref. 21), with the chemical half-life of the Dha₅ residue increasingnearly 60-fold, from less than a day to 48 days. Since the Nis₁₁₋₁₁-Sub₁₂₋₃₂ chimera has additional changes in the vicinity of the Dha₅residue, the chemical stability of the dehydro residues was examined bytaking the NMR spectrum of a sample that was incubated in aqueoussolution for an extended period of time.

A 3 mg amount of the Nis₁₋₁₁ -Sub₁₂₋₃₂ chimera (consisting of a mixtureof the Early Peak and Late Peak as defined in FIGS. 4 and 5) wasdissolved in D₂ O at a pH of 6.0, placed in an NMR tube (which was thenclosed), and incubated in aqueous solution in the dark at roomtemperature for 2.5 months. The NMR spectrum of this sample wasdetermined after 0, 34, and 72 days, with the results shown in FIG. 6.The NMR spectrum of the Nis₁₋₁₁ -Sub₁₂₋₃₂ chimera shows the expectedresonances of Dhb₂, Dhb₂ *, Dha₅, Dha₅ *, Dhb₈, and Dha₃₁. There was nosignificant change in the resonances of the dehydro residues during thecourse of the 72-day incubation period.

The slight differences that are seen are readily attributable tovariations introduced during baseline correction during computationswith the spectral data. In contrast to the 0.8-day half-life of the Dha₅residue in natural subtilin and its 48-day half-life in E4I-subtilin,the half-life of the Dha₅ residue in the Nis₁₋₁₁ Sub₁₂₋₃₂ chimera is solong that it cannot be estimated from the 72-day time-point. Longerincubation times were not performed. Therefore, the dehydro residues inthe Nis₁₁₋₁₁ -Sub₁₂₋₃₂ chimera are extremely stable.

These results demonstrate that the Dha₅ residue is subject to profoundchanges in its chemical reactivity, ranging from the most reactive stateobserved in natural subtilin to the least reactive state observed in theNis₁₋₁₁ -Sub₁₂₋₃₂ chimera, E4I-subtilin having an intermediate state ofreactivity. Somewhat surprisingly, the biological activity displayed bythese structural variants varies inversely with the reactivity, with theunstable and highly reactive subtilin having the lowest biologicalactivity, and the highly stable Nis₁₋₁₁ -Sub₁₂₋₃₂ chimera displaying thegreatest biological activity. The fact that the chemical reactivity ofDha₅ varies inversely with biological activity argues that role of theDha₅ residue in the antimicrobial mechanism is not related to itschemical reactivity in a simple fashion, and that other factors, such asthe specificity imposed by the peptide sequence surrounding the dehydroresidue, may also be important.

Properties of the Sub₁₋₁₁ -Nis₁₂₋₃₄ "Reverse Chimera". An importantfeature of the S_(L) -Nis₁₋₁₁ -Sub₁₂₋₃₂ chimeric prepeptide is that thesubtilin processing machinery is able to correctly recognize and processit into its corresponding mature form. Since the same machinery in B.subtilis does not successfully process S.sub. -Nis₁₋₃₄, there could besomething in the Nis₁₂₋₃₄ region that disturbs the subtilin processingmachinery of B. subtilis. If this is the case, the subtilin processingmachinery should not be able to correctly process a chimera thatcontains the Nis₁₂₋₃₄ region.

Accordingly, a reverse chimera was constructed (S_(L) -Sub₁₋₁₁-Nis₁₂₋₃₄), containing a subtilin sequence at the N-terminus of thestructural region and nisin sequence at the C-terminus. This chimera wasconstructed using the strategy described in FIG. 2. The synthetic genecontaining the "reverse" chimera was integrated into the chromosome ofLHermΔS and expressed. The corresponding polypeptide was recovered fromthe culture supernatant using the butanol-acetone extraction method, andfurther purified by RP-HPLC as shown in FIG. 7.

The HPLC profile of the Sub₁₁₋₁₁ -Nis₁₂₋₃₄ chimera is shown in thecenter of FIG. 7. A major peak emerged somewhat earlier than expectedfor the Sub₁₋₁₁ -Nis₁₂₋₃₄ chimera, but it was devoid of activity.Moreover, mass spectral analysis of the protein corresponding this majorpeak showed an Mr=3544.47 Da, which is about 56 mass units, or 3 watermolecules, greater than the expected 3488 Da. Thus, it could be thatthree dehydration reactions failed to occur in the processing of theprepeptide.

Following this large peak was a small peak (also shown in FIG. 7) thatshowed activity in the halo assay. The amount of material in this peakis quite small. Mass spectral analysis shows the material of the smallpeak to be very heterogeneous, consisting of at least a half-dozenspecies, none of which have the molecular mass expected for the Sub₁₋₁₁-Nis₁₂₋₃₄ chimera. Instead of an expected mass of 3488 Da, values of3079 (expected Mr-408; 13% of the total amount of peptides identifiedand analyzed in the minor peak), 3193 (Mr-295; 27%), 3322 (Mr-166; 12%),3437 (Mr-51; 27%), and 4174 (Mr+686; 21%) were obtained. None of thesemasses are readily explained in terms of simple processing defects, suchas the absence of dehydrations to give Dha₅ and Dha₃₃ (there is no Dhbexpected), or the leader peptide not being cleaved. The small size ofseveral species indicates that proteolysis may have occurred.

The halo assays of samples from the profile are shown at the bottom ofFIG. 7, with the arrows showing the samples which were derived from theprofile. The only activity in the profile corresponded to the positionof the small peak appearing at 16 min. The mass spectrum at the left wasobtained for the material in the large peak indicated by the arrow. Themass spectrum at the right was obtained for the material in the smallpeak (which was active) indicated by the other arrow.

The active specie(s) from production of the Sub₁₋₁₁ -Nis₁₂₋₃₄ chimera ina subtilin-producing microorganism have not yet been conclusivelydetermined. However, the specific activity of whatever specie(s) areresponsible for the inhibitory activity is much higher than nisinitself.

For example, the total area of the active peak consists of no more than10 μg of peptide, of which 0.13 μg was used for the halo assay shown inFIG. 7. This small quantity of peptide possesses an activity equivalentto 0.5 μg of nisin (data not shown). If all of the components in theminor peak were equally active, they would be about 4-fold (0.5 μg÷0.13)more active than nisin. The amount of the various components in theminor peak ranges from about 12% to about 27% of the total. If all ofthe activity is due to just one of the components, then the activecomponent would be about 15 to 35 times as active as nisin, depending onthe percentage of active component in the minor peak.

Determining the active species and the activities thereof will requirethat the active component be purified to homogeneity and studiedfurther. This can be accomplished by the procedures described herein(for example, by HPLC using a shallower gradient, as described above forseparation of succinylated Nis₁₋₁₁ -Sub₁₂₋₃₂, and by determiningbiological activity as described above). Although we do not conclusivelyknow which factors contribute to this high activity, the discovery ofthis high activity is completely unexpected, and will lead to the designof lantibiotic analogs with superior antimicrobial properties.

DISCUSSION

The ability to incorporate the unusual dehydro and lanthio-type aminoacids into lantibiotic analogs and non-lantibiotic polypeptides dependson the ability of the lantibiotic processing machinery to cope withforeign precursor sequences. Our working hypothesis is that the leaderregion is primarily responsible for engaging the prepeptide with theprocessing machinery, and once engaged, serines and threonines aredehydrated with little regard for the sequence in which they reside.Cysteines then react with particular dehydro residues in accordance withthe forces of folding and conformation that exist within the polypeptidein a manner that is reminiscent of the specific selection ofdisulfide-bond partners in polypeptides such as ribonuclease A andinsulin (33). There are now several known instances in whichpre-lantibiotic peptides undergo processing reactions, but give rise toinactive products. These instances are summarized in Table 1. Examplesinclude the S_(L) -Nis₁₋₃₄ chimera that produces a processed (22) butinactive (22, 23) product when expressed in a cell that possesses thesubtilin machinery, and the present S_(L) -Sub₁₋₁₁ -Nis₁₂₋₃₄ chimerathat produces a heterogeneous mixture of products that are mainlyinactive, although at least one active form is produced.

Although N_(L) -Nis₁₋₃₄ is an authentic lantibiotic precursor, thesubtilin processing machinery seems incapable of processing it, and itsgene products have not been detected in B. subtilis (22, 23). However,if the subtilin leader is placed in front of the nisin structural regionto give S_(L) -Nis₁₋₃₄, a processed, but inactive, product is producedby the subtilin machinery of B. subtilis (22). Thus, the subtilin leaderis competent in engaging the B. subtilis processing machinery, but thereis something about the conformational and folding interactions betweenthe leader and structural region in the S_(L) -Nis₁₋₃₄ construct thatcauses some of the processing reactions (perhaps the "partner" selectionin thioether formation) to malfunction. The fact that the S_(L)(1-7)-N_(L)(8-23) -Nis₁₋₃₄ construct is processed properly to give activenisin (23) argues that critical conformational interactions are restoredwhen an appropriate N-terminal sequence element from the subtilin leaderregion is combined with a C-terminal sequence element of the nisinleader.

                                      TABLE 1    __________________________________________________________________________              Strain in   Peptide is secreted                                   Secreted    Prepeptide              which                   Prepeptide is                          into extracellular                                   peptide is    Sequence  expressed                   processed                          medium   active                                        Ref.    __________________________________________________________________________    S.sub.L -S.sub.1-32               B. subtilis                   yes    yes      yes  a              6633              B. subtilis                   yes    yes      yes  b              168    N.sub.L -Nis.sub.1-34               L. lactis                   yes    yes      yes  c              11454              B. subtilis                   no     no       na   d              6633              B. subtilis                   no     no       na   e              168    S.sub.L Nis.sub.1-34               L. lactis                   yes    yes      no   f    S.sub.L Nis.sub.1-34               B. subtilis                   yes    yes      no   g              6633    S.sub.L(1-7) -N.sub.L(8-23) -Nis.sub.1-34               B. subtilis                   yes    yes      yes  h              6633    S.sub.L -Nis.sub.1-11 -Sub.sub.12-32               B. subtilis              168  yes    yes      yes  This work    S.sub.L -Sub.sub.1-11 -Nis.sub.12-34               B. subtilis                   heterogeneous                          yes      partially                                        This work              168    __________________________________________________________________________     Refs: a. (18), b. (21), c. (17), d. (22, 23), e. unpublished, f. (24) g.     (22, 23), h. (23). "na" means not applicable.

However, this combination of leader sequence elements must beappropriately complemented by the structural region. Whereas the S_(L)-Nis₁₋₁₁ -Sub₁₂₋₃₂ construct is processed correctly, the S_(L) -Sub₁₋₁₁-Nis₁₂₋₃₄ construct is not. However, it is expected that determinationof three non-dehydrated serine and/or threonine residues in the majorproduct, subject to one or more appropriate enzyme(s) of the subtilinprocessing machinery (e.g., contacted with an appropriatesubtilin-producing microorganism) may lead to production of abiologically active lantibiotic. Moreover, the processing reactions forthe latter construct when expressed in B. subtilis give a complexmixture of mainly inactive products.

Surprisingly, at least one component in this mixture of product isactive. None of the components of the S_(L) -Sub₁₋₁₁ -Nis₁₂₋₃₄ productmixture had the mass of a correctly-processed product, however.Therefore, the activity of the minor product(s) must be due to anincorrectly-processed component. Quite surprisingly, the specificactivity of the active component of the minor product(s) was at least4-fold and as much as 35-fold higher than nisin itself. The knowledgeabout what is responsible for such high activity may provide insightabout the design of lantibiotics which are dramatically more effectivethan the natural forms.

In conclusion, correct processing of the pre-lantibiotic peptide mayrequire specific conformational communication between the N-terminalportion of the leader region and the C-terminal portion of thestructural region of certain constructs. The results herein also providenew insight about the relationship between the structure of lantibioticsand their chemical properties and biological activity. Subtilin andnisin are highly disparate in their chemical stability and specificactivity, with nisin being superior to subtilin in both categories. TheNis₁₋₁₁ -Sub₁₂₋₃₂ chimera has the superior properties of nisin, showingthat the three residues that differ in the N-terminal regions of nisinand subtilin are primarily responsible for the disparity between nisinand subtilin.

Nis₁₋₁₁ -Sub₁₂₋₃₂ has a very hydrophobic N-terminal region, which mayfacilitate insertion of the lantibiotic into the membrane, which is itstarget of action (26, 34-36). However, another possible explanation forthe elevated activity of nisin the presence of a second dehydro residue(Dhb) at position 2 in the Nis₁₋₁₁ -Sub₁₂₋₃₂ chimera. One might expectthat the Dhb₂ would have a dramatic effect on the antimicrobialproperties of the chimera, since it is so close to the critical Dha₅ andmight cooperate in reacting with its microbial target. However, even ifDhb₂ does affect the antimicrobial properties, there may be no more thana 2-fold effect.

This illustrates a frustrating aspect of our knowledge aboutlantibiotics. The ubiquitous occurrence of the unusual residues amongthe many known lantibiotics argues that they are conserved because theyhave important functions. However, except for the critical role of Dha₅in inhibition of spore outgrowth, functions that clearly justify thisubiquitous occurrence have yet to be fully elucidated.

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

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31. Liu, W., and Hansen, J. N. (1993) Appl. Environ. Microbiol. 59,648-651.

32. Liu, W., and Hansen, J. N. (1990) Appl. Environ. Microbiol. 56,2551-2558.

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34. Kordel, M., Schuller, F., and Sahl, H.-G. (1989) FEBS Lett. 244,99-102.

35. Schuller, F., Benz, R., and Sahl, H.-G. (1989) Eur. J. Biochem. 182,181-186.

36. Benz, R., Jung, G., and Sahl, H.-G. (1991) in Nisin and NovelLantibiotics (Jung, G., and Sahl, H.-G., eds.), pp. 359-372, ESCOM,Leiden, The Netherlands.

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    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 12    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 34 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 2..3    (D) OTHER INFORMATION: /product="OTHER"    /note= "Dehydrobutyrine (Dhb)"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 3..7    (D) OTHER INFORMATION: /product="OTHER"    /note= "Positions 3 and 7 are lanthionine."    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 5..6    (D) OTHER INFORMATION: /product="OTHER"    /note= "Dehydroalanine (Dha)"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 8..11    (D) OTHER INFORMATION: /product="OTHER"    /note= "Positions 8 and 11 are beta-methyllanthionine."    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 13..19    (D) OTHER INFORMATION: /product="OTHER"    /note= "Positions 13 and 19 are beta-methyllanthionine."    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 23..26    (D) OTHER INFORMATION: /product="OTHER"    /note= "Positions 23 and 26 are beta-methyllanthionine."    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 25..28    (D) OTHER INFORMATION: /product="OTHER"    /note= "Positions 25 and 28 are beta-methyllanthionine."    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 33..34    (D) OTHER INFORMATION: /product="OTHER"    /note= "Dehydroalanine (Dha)"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    IleXaaXaaIleXaaLeuXaaXaaProGlyXaaLysXaaGlyAlaLeu    151015    MetGlyXaaAsnMetLysXaaAlaXaaXaaHisXaaSerIleHisVal    202530    XaaLys    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 32 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 3..7    (D) OTHER INFORMATION: /product="OTHER"    /note= "Positions 3 and 7 are lanthionine."    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 5..6    (D) OTHER INFORMATION: /product="OTHER"    /note= "Dehydroalanine (Dha)"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 8..11    (D) OTHER INFORMATION: /product="OTHER"    /note= "Positions 8 and 11 are beta-methyllanthionine."    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 13..19    (D) OTHER INFORMATION: /product="OTHER"    /note= "Positions 13 and 19 are beta-methyllanthionine."    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 18..19    (D) OTHER INFORMATION: /product="OTHER"    /note= "Dehydrobutyrine (Dhb)"    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 23..26    (D) OTHER INFORMATION: /product="OTHER"    /note= "Positions 23 and 26 are beta-methyllanthionine."    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 25..28    (D) OTHER INFORMATION: /product="OTHER"    /note= "Positions 25 and 28 are beta-methyllanthionine."    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 31..32    (D) OTHER INFORMATION: /product="OTHER"    /note= "Dehydroalanine (Dha)"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    TrpLysXaaGluXaaLeuXaaXaaProGlyXaaValXaaGlyAlaLeu    151015    GlnXaaXaaPheLeuGlnXaaLeuXaaXaaAsnXaaLysIleXaaLys    202530    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 13 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 3..7    (D) OTHER INFORMATION: /product="OTHER"    /note= "Positions 3 and 7 are lanthionine."    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 8..11    (D) OTHER INFORMATION: /product="OTHER"    /note= "Positions 8 and 11 are beta-methyllanthionine."    (ix) FEATURE:    (A) NAME/KEY: Peptide    (B) LOCATION: 13    (D) OTHER INFORMATION: /note= "Position 13 represents    either positions 13-34 of Nisin or positions 13-32 of    subtilin."    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    XaaXaaXaaXaaXaaLeuXaaXaaProGlyXaaXaaXaa    1510    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 127 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: unknown    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid    (A) DESCRIPTION: /desc = "Synthetic DNA"    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..117    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    GATTCGAAAATCACTCCGCAAATCACTAGTATTTCACTTTGTACACCC48    AspSerLysIleThrProGlnIleThrSerIleSerLeuCysThrPro    151015    GGGTGTGTAACTGGTGCATTGCAAACTTGCTTCCTTCAAACACTAACT96    GlyCysValThrGlyAlaLeuGlnThrCysPheLeuGlnThrLeuThr    202530    TGTAACTGCAAAATCTCTAAATAGGTAACCC127    CysAsnCysLysIleSerLys    35    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 39 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    AspSerLysIleThrProGlnIleThrSerIleSerLeuCysThrPro    151015    GlyCysValThrGlyAlaLeuGlnThrCysPheLeuGlnThrLeuThr    202530    CysAsnCysLysIleSerLys    35    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 38 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid    (A) DESCRIPTION: /desc = "Synthetic DNA"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    TGAATTCAGATTCGAAAATCACTCCGCAAATCACTAGT38    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 49 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid    (A) DESCRIPTION: /desc = "Synthetic DNA"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    ACCAAAGCTTCAACCCGGGTGTACAAAGTGAAATACTAGTGATTTGCGG49    (2) INFORMATION FOR SEQ ID NO:8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 144 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: unknown    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid    (A) DESCRIPTION: /desc = "Synthetic DNA"    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..123    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    GATTCGAAAATCACTCCGCAATGGAAAAGTGAATCACCTTGTACACCC48    AspSerLysIleThrProGlnTrpLysSerGluSerProCysThrPro    40455055    GGGTGTAAAACCGGCGCCCTGATGGGTTGTAACATGAAAACAGCCACG96    GlyCysLysThrGlyAlaLeuMetGlyCysAsnMetLysThrAlaThr    606570    TGTCATTGTAGTATTCACGTAAGCAAATAGGTAACCAAATAGGTAAC143    CysHisCysSerIleHisValSerLys    7580    C144    (2) INFORMATION FOR SEQ ID NO:9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 41 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    AspSerLysIleThrProGlnTrpLysSerGluSerProCysThrPro    151015    GlyCysLysThrGlyAlaLeuMetGlyCysAsnMetLysThrAlaThr    202530    CysHisCysSerIleHisValSerLys    3540    (2) INFORMATION FOR SEQ ID NO:10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 35 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid    (A) DESCRIPTION: /desc = "Synthetic DNA"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    GAGAATTCTATCCCGGGTGTAAAACCGGCGCCCTG35    (2) INFORMATION FOR SEQ ID NO:11:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 36 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid    (A) DESCRIPTION: /desc = "Synthetic DNA"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    ATGGGTTGTAACATGAAAACAGCCACGTGTCATTGT36    (2) INFORMATION FOR SEQ ID NO:12:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 54 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid    (A) DESCRIPTION: /desc = "Synthetic DNA"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    GTGAAAGCTTGGGGTTACCTATTTGCTTACGTGAATACTACAATGACACGTGGC54    __________________________________________________________________________

What is claimed as new and desired to be secured by Letters Patent ofthe United States is:
 1. A mutant or chimeric lantibiotic of theformula: ##STR2## wherein Z is Sub₁₃₋₃₂,Xaa at the 1-position is Trp,Phe or Tyr; Xaa at the 2-position is Lys, Arg or His; Xaa at the4-position is Ile, Gly, Ala, Val, or Leu; Xaa at the 5-position is Dha;and Xaa at the 12-position is Val, Ile, Gly, Ala or Leu, and whereinsaid lantibiotic has antimicrobial activity, with the proviso that,simultaneously, the 1-position, the 2-position, the 4 position and the5-position are not, respectively, Trp, Lys, Ile, and Dha or Ala.
 2. Themutant or chimeric lantibiotic of claim 1, where Xaa at the 1-positionis Trp; Xaa at the 2-position is Lys; Xaa at the 4-position is Gly, Ala,Val, or Leu; and Xaa at the 12-position is Val.
 3. The mutant orchimeric lantibiotic of claim 1, where Xaa at the 1-position is Trp; Xaaat the 2-position is Lys; Xaa at the 4-position is Ile; Xaa at the5-position is Dha; and Xaa at the 12-position is Val, subject to saidproviso.
 4. The mutant or chimeric lantibiotic of claim 1, having thesequence Nis₁₋₁₁ -Sub₁₂₋₃₂.
 5. The mutant or chimeric lantibiotic ofclaim 1 produced by a process comprising:culturing, in a suitableculture medium, a lantibiotic-producing host transformed with (1) apolynucleic acid encoding the lantibiotic, (2) an expression vectorcomprising the polynucleic acid or (3) a plasmid comprising thepolynucleic acid; and recovering the mutant or chimeric lantibiotic fromthe culture medium.
 6. The mutant or chimeric lantibiotic of claim 1,having an antimicrobial activity at least equal to that of nisin.